Many multimeric bacterial toxins that comprise monomers or subunits that comprise monomers are known in the art. These include multimeric pore-forming toxins which lack a second catalytic effector domain or molecule, and multimeric binary toxins which comprise a second catalytic effector domain or molecule. Staphylococcal α-hemolysin, Staphylococcal leukocidin, aerolysin (e.g., from Aeromonas hydrophila), Clostridium septicum α toxin, Bacillus cereus hemolysis II, and Helicobacter pylori vacuolating toxin (VacA) are examples of multimeric pore-forming toxins which lack a second catalytic effector domain. Anthrax toxin, cholera toxin, E. coli heat-labile enterotoxin, Shiga toxin, pertussis toxin, Clostridium perfringens iota toxin, Clostridium spiroforme toxin, Clostridium difficile binary toxin, Clostridium botulinum C2 toxin, and Bacillus cereus vegetative insecticidal protein are examples of multimeric binary toxins which comprise a second catalytic effector domain or molecule. The interaction between catalytic effector domain of these toxins and target cells leads to the toxic effects of these toxins. For example, anthrax toxin lethal factor (LF), cholera toxin subunit A, Shiga toxin subunit A, C. perfringens iota toxin 1 a component 1 a (an ADP-ribosyltranferase), C. spiroforme toxin subunit A, C. difficile toxin subunit A, a C. botulinum C2 subunit A; and B. cereus vegetative insecticidal protein subunit A each serve as the catalytic effector domain of their respective toxins.
Homo-oligomeric bacterial toxins with modified monomers have been designed and used to target particular cell populations. For example, Liu et al., PNAS USA 100(2):657-662 (2003) describe use of a modified homo-oligomeric anthrax toxin protective antigen (PrAg) in which the native furin cleavage site has been replaced by a urokinase plasminogen activator cleavage site to target and kill melanoma cells, fibrosarcoma cells, and lung carcinoma cells in vivo. Liu et al., J. Biol. Chem., 276(21): 17976-17984 (2001) describe use of a modified homo-oligomeric PrAg in which the native furin cleavage site has been replaced by a urokinase plasminogen activator cleavage site to target and kill melanoma cells, adenocarcinoma cells, and lung carcinoma cells in vitro. Liu et al., Cancer Res. 60:6061-6067 (2000) and WO 01/21656 describe use of a modified homo-oligomeric PrAg in which the native furin cleavage site has been replaced by a matrix metalloproteinase cleavage site (e.g., for MMP-2 or MMP-9) to target and kill melanoma cells, fibrosarcoma cells, and breast cancer cells in vitro. These homo-oligomeric PrAg comprising modified monomers are also described in Liu et al., Expert Opin. Biol. Ther. 3(5):843-853 (2003). U.S. Pat. No. 5,677,274 describes, inter alia, use of a modified PrAg in which the native trypsin cleavage site has been replaced by a cleavage site for HIV-1 protease to target and kill HIV-infected cells in vitro. WO 03/033648 describes use of a modified anthrax toxin protective antigen (PrAg) in which the native furin cleavage site has been replaced by a matrix metalloproteinase cleavage site or a plasminogen activator cleavage site to target and detect, i.e., image, target cells expressing matrix metalloproteinases or plasminogen activators on their surface.
Hetero-oligomeric bacterial toxins based on binary bacterial toxins have also been designed. These toxins have been used to explore the interactions between one component of the binary toxins (e.g., the binding component and the catalytic effector component) as well as the interactions between the monomers themselves. Mogridge et al., PNAS USA 99(10):7045-7048 (2002) describe a modified hetero-oligomeric PrAg comprising two types of modified monomers in which the monomer binding sites have been mutated so that the two types of monomers can only form oligomers with each other. Cunningham et al., PNAS USA 99(10):7049-7053 (2002) describe a modified hetero-oligomeric PrAg comprising two types of modified monomers in which the LF binding sites have been modified so that both types of monomers are required to bind LF.
Thus, bacterial toxins have been used in the development of homo-oligomeric and hetero-oligomeric toxins. The homo-oligomeric toxins, in particular, have been used to target specific cell populations (e.g., cancer cells or virally infected cells). The monomers of the homo-oligomeric toxins were modified to take advantage of a single characteristic of the target cell population. More particularly, the monomers were modified to replace a native proteolytic cleavage site with a cleavage site for a cell surface protease (e.g., MMP or plasminogen activator) overexpressed in the target cells, and thus specifically target these cells. The homo-oligomeric toxins can sometimes target normal cells which share the single characteristic of the target cell population. Therefore, hetero-oligomeric toxins which rely on multiple characteristics of a cell population are likely to have increased target cell specificity and decreased non-specific toxicity to non-target cells.
Thus, there is a need in the art for additional modified bacterial toxins which have greater specificity for a particular target cell population, i.e., toxins that target cells based on more than one target cell characteristic, and methods of using such toxins. The present invention addresses these and other needs.